Hindawi Publishing Corporation Journal of Toxicology Volume 2016, Article ID 9794570, 7 pages http://dx.doi.org/10.1155/2016/9794570

Research Article Evaluation of (Dream Herb,) zacatechichi,for Nephrotoxicity Using Human Kidney Proximal Tubule Cells

Miriam E. Mossoba, Thomas J. Flynn, Sanah Vohra, Paddy Wiesenfeld, and Robert L. Sprando Center for Food Safety and Applied Nutrition (CFSAN), Office of Applied Research and Safety Assessment (OARSA), Division of Toxicology (DOT), US Food and Administration (US FDA), Neurotoxicology and In Vitro Toxicology Branch (NIVTB), 8301 Muirkirk Road, Laurel, MD 20708, USA

Correspondence should be addressed to Miriam E. Mossoba; [email protected]

Received 29 February 2016; Accepted 1 August 2016

Academic Editor: Orish Ebere Orisakwe

Copyright © 2016 Miriam E. Mossoba et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,” Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening of C. zacatechichi forsafetyespeciallyimportant.Weexposedhuman proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects of C. zacatechichi relative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support forthe need to further scrutinize the safety of this herbal dietary supplement.

1. Introduction expression [11–13]. In addition, germacrolides are part of the class of sesquiterpene lactones, which can also exhibit Calea zacatechichi (also called Calea ternifolia or “Dream negative effects on both prokaryotic and mammalian cells Herb”) is a flowering native to Central America and [14]. The cytotoxicity of both flavonoids and sesquiterpene has a long tradition of use as a medicinal plant in indige- lactones has been exploited for use as therapy against cancer nous cultures [1]. Exposure through inhalation (smoking) or [15, 16]. ingestion (as tea) is primarily used to temporarily intensify Despite clear evidence that at least some of the biologi- lucid dreaming. It is also widely consumed to treat problems cally active components of C. zacatechichi have the potential associated with the gastrointestinal and endocrine systems to be cytotoxic, safety evaluations of whole forms of this [2]. It has recently been marketed as a dietary supplement herbal supplement are lacking, especially ones that focus on in the management of diabetes due to its ability to induce the kidney. The kidneys use a complex transport system to hypoglycemic effects [3–5] although its mechanism(s) of eliminate unwanted chemicals, regulate blood pressure and action remain unclear. glucose levels, and maintain a balanced pH [17]. However, as The oneirogenic and other biological effects of C. zacat- the glomerular filtrate passes through the tubular system, the echichi are attributed in part to their flavones and germa- reabsorption of water and electrolytes by the proximal tubule crolides components [6–10]. However, flavones represent a cells can progressively concentrate chemicals in the lumen class of flavonoids that have been shown to carry cytotoxic that do not get reabsorbed. Unfortunately, the proximal effects in part through induction of cytochrome P450 enzyme tubules can become exposed to toxic concentrations of such 2 Journal of Toxicology chemicals, even when blood concentrations are relatively processes. Treated cells in black-wall, clear bottom 96-well lower, leaving the kidneys vulnerable to injury [17]. In the plates were equilibrated to room temperature for 30 minutes, case of C. zacatechichi,itisunknownwhetheranyofits during which time water in the outer wells was replaced with componentscanbenephrotoxic,butgiventhatitismarketed approximately 100 uL of treatment or media only controls. to diabetics, any preexisting diabetic nephropathy marked Following that, an equal volume of CellTiter-Glo working by glomerular or proximal tubule damage [18–20] could solution was added to each well. Plates were placed on induce further kidney damage. Therefore, we focused our an orbital shaker for 2 minutes to induce cell lysis and research on screening for the potential nephrotoxicity of then incubated for an additional 10 minutes before being C. zacatechichi using an in vitro model of human proximal read on an OMG Fluorostar plate reader (BMG LABTECH, tubule cells. We chose the HK-2 cell line as our human Ortenberg, Germany) to measure the levels of luminescence proximal tubule model for its robust performance in many emitted from each well. in vitro toxicology studies [21–25]. We compared the effects of exposing HK-2 cells to C. zacatechichi and two control 2.4. Reactive Oxygen Species Assay. Quantification of reactive compounds, a known renal toxicant (cisplatin) and a known oxygen species (ROS) was determined using Promega’s ROS- renal protectant (valproic acid), and evaluated their dose- Glo H2O2 luminescence-based detection system and data dependent effects on cytotoxicity, mitochondrial injury, and were normalized to cell viability. Following 24 hrs of direct four kidney-specific biomarkers of toxicity [26–28]: (1) Kid- exposure to C. zacatechichi, cells were incubated with H2O2 ney Injury Molecule-1 (KIM-1), (2) Albumin, (3) Cystatin substrate and detection reagent, as recommended in the 𝛽 C, and (4) 2-microglobulin (B2M). KIM-1 is expressed manufacturer’s instructions. Luminescence was read on an in tubular epithelial cells in response to injury. Albumin, OMG Fluorostar plate reader. Cystatin C, and B2M are indicators of impaired reabsorption by the proximal tubules. In this study, we demonstrate that 2.5. Mitochondrial Membrane Potential Assay. Changes in C. zacatechichi is capable of inducing both cellular and mitochondrial membrane potential (MMP) were evaluated organellar toxicity in proximal tubule cells. using the ratiometric dye JC-10 (Enzo, Farmingdale, NY). HK-2 cells that were directly exposed to C. zacatechichi were 2. Materials and Methods stained with 20 uM JC-10 (final concentration) for 2 hours, washed, and then read by plate reader (OMG Fluorostar). 2.1. Characterization of Calea zacatechichi Extract. Voucher Excitation was set at 485 nm and emission at 520 and 590 nm samples of C. zacatechichi deposited at the University of was measured. We also verified that extract or media alone Mississippi, National Center for Natural Products Research did not produce significant emission signals. (NCNPR) (NCNPR #2443), were authenticated using macroscopy and microscopy methods by an NCNPR 2.6. Nephrotoxicity Biomarker Assays. Culture supernatants botanist. A methanol-extract of C. zacatechichi was provided from cells treated for 24 hours with C. zacatechichi,cisplatin, in lyophilized form by NCNPR and was stored in the dark at ∘ andvalproicacidatdosesof333and111𝜇g/mL were evaluated 4 C in a vacuum chamber. Dried extract of C. zacatechichi forlevelsofbiomarkersofkidneytoxicity:KidneyInjury- was analyzed by LC/QTof as described previously [29]. 1 (KIM-1), Albumin, Cystatin C, and beta-2-microglobulin Compounds were putatively identified by matching exact (B2M) using the Human Kidney Toxicity kits (Bio-Rad, mass of analytes with components of C. zacatechichi reported Hercules, CA). Following the manufacturer’s protocol, plates in the literature [8, 30–33]. wereblocked,washed,andincubatedwithsamples,standard solutions detection antibodies, before being given a final 2.2. Cell Culture and Treatments. HK-2 cells were grown, wash. Plates were read using a Luminex 200 instrument (Bio- maintained, and treated in a manner similar to that described Rad). Biomarker expression levels were normalized to cell previously [29]. Stock treatment solutions of C. zacatechichi, viability. nephrotoxicant (positive control) cis-diamineplatinum(II) dichloride (cisplatin) (Sigma-Aldrich, St. Louis, MO), and 2.7. Statistics. Microsoft Excel and Prism (GraphPad, San nephroprotectant (negative control) valproic acid (Sigma- Diego, CA) were used for calculations and analyses of all Aldrich) were made by weighing out their powders, dissolv- data collected. Student’s 𝑡-tests or 2-way ANOVAs were used ing them in DMSO, and diluting this mixture with media to determine whether dose-matched treatment effects were for a final DMSO stock solution of 0.4% or less. Cells were statistically significant at 𝑃 values less than 0.01 or 0.001 as incubated overnight and treated in triplicate for 24 hours at indicated. the dose range of 0–1000 𝜇g/mL. 3. Results 2.3. Cytotoxicity Assay. Treatment-related cytotoxicity was determined using the established CellTiter-Glo Cell Viability 3.1. Characterization of Calea zacatechichi Extract. LC-high Assay (Promega, Madison, WI) following the manufacturer’s resolution MS found 231 total molecular features in the C. recommendations. The premise of this luminescent assay is zacatechichi extract. Of these, 24 features had exact mass con- that ATP production is directly proportional to cell viabil- sistent with that of reported components of C. zacatechichi ity, as ATP is central to energy required for vital cellular (Figure 1). The major components based on peak volume, Journal of Toxicology 3

1.4E + 06 200000 6 HK-2 cell viability 1.2E + 06

1.0E + 06 4 ∗ ∗ 150000 ∗ 8.0E + 05 ∗ 6.0E + 05 Counts 100000 7 4.0E + 05 2 89 ∗ 1 2.0E + 05 5 10 3 ATP luminescence ATP 50000 ∗ 0.0E + 00 012345678910 ∗ RT (min) 0 Figure 1: Extracted total compound chromatogram from chemical 0 50 100 150 200 1000 characterization of the C. zacatechichi extract by LC-high resolution Treatment concentration (𝜇g/mL) mass spectroscopy. Putative compound identification was made by matching exact mass with that of known components of C. Figure 2: C. zacatechichi significantly decreases cell viability ina zacatechichi [8, 30–33]. (1) Ciliarin, (2) zexbrevin, (3) sesquiterpene dose-dependent manner. HK-2 cells were treated with C. zacate- lactone, (4) calein D, (5) 1-𝛽-acetoxyzacatechinolide, (6) calein A, chichi (open circles), cisplatin (open squares), or valproic acid (filled ± (7) 1-oxo-zacatechinolide, (8) calealactone E, (9) calealactone, and circles) at mean average concentrations ( SEM) ranging from 0 𝜇 (10) acetoxycaleculatolide. to 1000 g/mLandcellviabilitywasquantitativelyassayedbyATP luminescence 24 hours after treatment. Dashed line indicates “no treatment” baseline ATP levels. ∗, cisplatin or valproic acid versus C. zacatechichi, 𝑃 < 0.001. calein A, ciliarin, acacetin, and calealactone C, accounted for about 50% of the known compounds and 8% of the total compounds [8, 30–33]. created in the intracellular environment of HK-2 cells. Using a luminescence assay of ROS detection, we found that C. 3.2. C. zacatechichi Strongly Inhibits HK-2 Cell Viability. To zacatechichi treatment led to increasingly higher levels of ROS investigate the nephrotoxicity of C. zacatechichi,weper- production in a manner that was directly proportional to formedanATP-basedcellviabilityassayonHK-2cellstreated the increasing treatment dose (Figure 3(a)). ROS production 𝜇 with a 6-dose concentration range from 0 to 1000 g/mL for from cells treated with up to 333 𝜇g/mL of C. zacatechichi was 24 hours. For comparison, we also treated cells for 24 hours intermediary between those from the positive- and negative- with dose-matched concentrations of the known nephro- control treated cells. At the 1000 𝜇g/mL dose, however, C. toxic compound, cisplatin, and the known nephroprotectant, zacatechichi induced ROS levels that surpassed those in valproic acid. We found that cisplatin induced a significant cisplatin-treated cells (𝑃 < 0.001), as shown in Figure 3(a). ∼ 𝜇 reduction in cell viability starting at the 12 g/mL dose To gain a better understanding of (1) whether the elevated 𝑃 < 0.001 tested ( ) and caused complete cell death at the maxi- levels of ROS production actually correlated with mitochon- mum dose tested (Figure 2). Similarly, significant cytotoxicity drial injury and (2) to what extent injury took place, we 𝜇 𝑃< of C. zacatechichi was detected starting at 37.0 g/mL ( performed a ratiometric assay using JC-10 dye to compare 0.001 𝜇 )andstillachievedcompletecelldeathby1000 g/mL. the relative levels of damaged and healthy mitochondria in For the range of doses tested, the cytotoxic effect of C. treated HK-2 cells. Compared to the baseline ratio value of zacatechichi was directly proportional to the treatment dose about 5, treatment with C. zacatechichi led to a uniquely sharp and we calculated its lethal concentration 50 (LC50)value increase in the ratio of damaged to healthy mitochondria to be 91.7 𝜇g/mL, compared to 13.3 𝜇g/mL for cisplatin. By starting from the 12.3 𝜇g/mL testing dose and achieved a contrast, valproic acid successfully maintained cell viability maximum ratio value of about 50 when the treatment dose across the range of tested doses, except for the maximum was increased to just 37.0 𝜇g/mL (Figure 3(b)). This maxi- dose of 1000 𝜇g/mL, where cell viability dropped only slightly, mal relative level of mitochondrial damage was statistically asshowninFigure2.Asexpectedfromanephroprotectant, significant𝑃 ( < 0.001)andwaswellsustainedforthe the calculated LC50 value of valproic acid is quite high at remaining higher treatment doses of C. zacatechichi.By 3866 𝜇g/mL, given the plateau shape of its cell viability curve. contrast, cisplatin induced mitochondrial damage at a much slower rate to achieve a ratio value of ∼50 at 333 𝜇g/mL. As expected, the mitochondrial injurious effects from valproic 3.3. Mitochondrial Toxicity Increases Proportionately with acid were minimal over the spectrum of treatment doses. Higher Exposure to C. zacatechichi. To begin studying early events of cellular toxicity, we evaluated how the mitochondria 3.4. Proximal Tubule Cell Function Is Significantly Com- of HK-2 cells were affected by 24-hour treatments with C. promised by C. zacatechichi. To address whether renal cell zacatechichi relative to treatments with cisplatin or valproic function would become compromised after treatment with C. acid. We first measured the levels of ROS produced in zacatechichi, we evaluated cellular biomarkers that are strong treated cells to indicate the levels of oxidative stress that was indicators of nephrotoxicity [26–28]. We used a sensitive 4 Journal of Toxicology

108 Levels of ROS Levels of depolarized mitochondria 100 7 ∗ 10 ∗

106

5 ∗ 10 ∗ nm ratio 10 ∗

590 ∗ 104 ∗

3 nm : ∗

ROS luminescence ROS 10 520 2 10 1 101 0 50 100 150 200 1000 0 50 100 150 200 1000 Treatment concentration (𝜇g/mL) Treatment concentration (𝜇g/mL) (a) (b)

Figure 3: Cellular stress induced by C. zacatechichi is indicated by a surge in ROS and a rapid shift toward MMP loss. HK-2 cells treated for 24 hours with C. zacatechichi (open circles), cisplatin (open squares), or valproic acid (filled circles) were assayed for cellular levels of (a) normalized mean average ROS levels (± SEM) as well as (b) changes in the relative amounts of mitochondria that undergo loss versus maintenance of membrane potential, calculated as a mean average (± SEM) ratio of fluorescence emission at 520 versus 590 nm. ∗,cisplatin or valproic acid versus C. zacatechichi, 𝑃 < 0.001.

multiplex approach to simultaneously detect differences in on the mechanism(s) of toxicity [37]. We chose to use the levels of four FDA-qualified biomarkers: KIM-1, Albu- thehumanrenalproximaltubuleepithelialcellline,HK-2, min,CystatinC,andB2M.Wequantitatedtheconcentrations because the proximal tubule plays a critical role in controlling oftheseanalytesintheculturesupernatantsofHK-2cells the clearance and reabsorption processes of xenobiotics and exposed to 111 or 333 𝜇g/mL of C. zacatechichi,cisplatin, their metabolites [38, 39]. Proximal tubule epithelial cells valproic acid, or untreated media for 24 hours (Figure 4). In encounter toxicants that are filtered and are an important agreement with our findings of cellular and mitochondrial componentofoverallnephrotoxicitythatcanleadtoboth toxicity, we found significantly elevated levels for nearly all acute and chronic kidney damage [40]. The HK-2 cell line is of these markers (𝑃 < 0.01)inculturesupernatantsofHK-2 an appropriate choice for establishing a renal cell toxicology cells treated with C. zacatechichi compared to those treated profile on C. zacatechichi for two main reasons. First, HK-2 with valproic acid or left untreated. The extent of biomarker cells are human derived and thus, data generated from this elevation induced by C. zacatechichi never exceeded that of cell line are not confounded by differences between human cisplatin. This trend was also observed at the lower treatment and other species. Second, HK-2 cells closely recapitulate dose of 111 𝜇g/mL, even though the actual concentrations of many aspects of the morphological and metabolic phenotype each biomarker were typically 10-fold less than in high-dose of proximal tubule cells in vivo [23, 41]. In our evaluation treatments, as shown in Figure 4. of cytotoxicity, we found striking similarities between our tested herbal extract and cisplatin at single dose treatment 4. Discussion concentrations as low as approximately 37 𝜇g/mL in the form of short-term exposure. Moreover, it appeared that the Although C. zacatechichi is not a controlled substance under mechanism of action of C. zacatechichi’s active ingredients federal law, it has been banned in the state of or renal-derived metabolites resembled those of the highly LouisianaaswellasinPolandonthebasisofitsmind-altering injurious cisplatin; elevated ROS levels and a severe loss effects [34, 35]. In our study, we used an in vitro human of mitochondrial membrane potential were hallmarks of renal proximal tubule cell model to perform several assays nephrotoxicity shared by these two substances. By contrast, that collectively evaluated the nephrotoxicity potential of C. valproicacidshowedlittleornotoxicitypotentialuntilthe zacatechichi. We used its extract to best model the highest dose of 1000 𝜇g/mL was tested. The relatively high tincture dietary supplements marketed in the United States. level of toxicity that was induced by C. zacatechichi within By comparing its toxicity profile to that of a highly toxic pure the 24 hours of direct exposure to HK-2 cells is of concern. compound, cisplatin, and an innocuous pure compound, However, since no data exist on the serum concentrations valproic acid, we established a stringent in vitro cell culture of C. zacatechichi’s active components, it is unclear how our safety evaluation model system. Although we identified chosen treatment doses compare to what kidney cells in vivo several of the chemical components of C. zacatechichi,we would be exposed to, especially postmetabolism by the gut were focused on evaluating the toxicity of this herbal extract and liver. as a whole. In vitro testing not only provides a window Although further studies would be needed to elucidate a into cell-specific effects [36] but also yields informative data more detailed mechanistic analysis of C. zacatechichi’s modes Journal of Toxicology 5

100 1000 Albumin KIM-1 ∗

∗ 100 10 ∗ 10

∗ ∗ ∗ ∗ 1 concentration (ng/mL) concentration

1 1 KIM- Albumin concentration (ng/mL) concentration Albumin 0.1 0.1 CZ CIS VAL NT CZ CIS VAL NT Treatment Treatment

1000 1000 B2M Cystatin C ∗ ∗ ∗ ∗ 100 100 ∗ ∗

10 ∗∗ 10 B2M concentration (ng/mL) B2M concentration Cystatin C concentration (ng/mL) C concentration Cystatin 1 1 CZ CIS VAL NT CZ CIS VAL NT Treatment Treatment Figure 4: Biomarker signature of C. zacatechichi-associated nephrotoxicity. HK-2 cells were treated with C. zacatechichi (CZ), cisplatin (CIS), or valproic acid (VAL) or left untreated (NT). Treatment concentrations were either 333 𝜇g/mL (black bars) or 111 𝜇g/mL (gray bars). At 24 hours after treatment, HK-2 cell culture supernatants were harvested and assayed by Bio-Plex assay for average levels of KIM-1, Albumin, Cystatin C, and B2M (± SEM). Biomarker expression levels were normalized to cell viability. ∗, cisplatin or valproic acid versus C. zacatechichi, 𝑃 < 0.01. of action, we have found additional evidence of its potential InsupportoftheideathatC. zacatechichi has the potential to cause significant renal cell damage. Specifically, our panel to cause cell injury, other groups have shown that extracts of indicators of kidney injury showed that not only were these of this herb or its purified components can exert inhibitory biomarkers elevated, but also the intensity of their elevation effects on cells using in vitro model systems. For example, approached that measured in our assays following cisplatin arecenttoxicologystudyhasshownthatC. zacatechichi treatment. The biomarkers we selected included those that can inhibit the transcription factor NF-kappaB, which is have been qualified by the FDA to serve as official biomarkers critical to regulating cellular inflammation and other func- of nephrotoxicity. They have gained attention recently as tions [45, 46]. A further understanding of C. zacatechichi’s theyhavebeenshowntobesolidcorrelatesofin vivo mechanism(s) of action may be extrapolated from studies on nephrotoxicity [42]. Overall, our findings indicated that the other members of the Calea genus. For example, C. platylepis, cellular toxicity of C. zacatechichi was capable of producing C. uniflora,andC. serrata havebeenshowntopossess elevations in all four biomarkers at the high treatment dose, but to a lesser extent than cisplatin. potent antimicrobial, antifungal, and acaricidal activities, Other toxicology studies on C. zacatechichi using in vivo respectively [47–49]. In addition, C. pinnatifida was shown model systems have not specifically evaluated nephrotoxicity to have cytotoxic effects against a wide variety of human endpoints but have still demonstrated its potential to have cell lines derived from a variety of organ systems, including a range of side effects. In a rat model, for example, extracts kidney [50]. Moreover, studies on germacrolides, which are of this herb were reported to inhibit edema and neutrophil common components of most herbs in the Calea genus, migration [43]. In a feline model, it caused ataxia, vomiting, including C. zacatechichi, have demonstrated the potential and unusual electroencephalogram (EEG) recordings [44]. for antileishmania effects [8], inhibition of cellular differen- In human volunteers, it resulted in significant increases in tiation [51], and cytotoxicity against human leukemia cells respiratory rates and decreases in reaction times [44]. [52, 53]. 6 Journal of Toxicology

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